Plasminogen activator inhibitors from placenta and fibrosarcoma cells are antigenically different as evaluated with monoclonal and polyclonal antibodies.

Plasminogen activator inhibitor (PAI) purified from human placenta was compared to PAI purified from conditioned cell culture fluid of the human fibrosarcoma cell line HT-1080. The two inhibitors had a similar mobility (Mr approximately 50,000) in sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Purified placental inhibitor revealed 2 major and 1 minor Coomassie blue stainable bands, while the fibrosarcoma inhibitor appeared as one band. By immunoblotting analysis both monoclonal and polyclonal antibodies against each of the inhibitors showed reaction with the inhibitor against which they were raised, but not cross reaction with the other inhibitor. Similar results were obtained, when antibody binding was tested by ELISA with the inhibitors coated on the solid phase. HPLC fingerprint patterns of cyanogen bromide fragments of the two inhibitors were different. The inhibitory activity of the placental PAI was decreased by a factor of 3 after incubation with SDS, while that of the fibrosarcoma PAI was increased by a factor of 30. It is concluded that the two inhibitors show no detectable common antigenic determinants and most likely are products of different genes.

Localization of plasminogen activators and plasminogen-activator inhibitors in human gingival tissues demonstrated by immunohistochemistry and in situ hybridization.

The plasminogen-activating system plays an important part in tissue proteolysis in physiological as well as pathological processes. Plasminogen activators u-PA (urokinase) and t-PA (tissue) as well as the inhibitors PAI-1 and PAI-2 are present in gingival crevicular fluid in concentrations significantly greater than in plasma. This fact, and the finding that the concentrations of t-PA and PAI-2 are higher in areas with gingival inflammation, indicate local production of these components. The present study describes, by means of in situ hybridization and immunohistochemistry, the localization of the plasminogen activators and their inhibitors in gingival tissues from patients undergoing periodontal surgery. t-PA mRNA and t-PA antigen were primarily found in the epithelial tissues, predominantly in the sulcular and junctional regions, although occasionally in the oral epithelium and in blood vessels of the connective tissue. u-PA and u-PA-receptor signals were seen in single cells within the junctional and sulcular epithelia and adjacent to blood vessels close to the junctional epithelium, but rarely in the oral epithelium. Similar to t-PA, the predominant location of PAI-2 mRNA was the gingival epithelia. In the junctional and sulcular epithelia, PAI-2 mRNA was seen throughout the thickness, while in the oral epithelium the strongest signals were seen in stratum granulosum and stratum spinosum. PAI-1 mRNA was invariably found in the connective tissue associated with blood vessels. The present study confirms earlier indications of local production of plasminogen activators and their inhibitors in gingival tissues. In addition, the results demonstrate that t-PA and PAI-2 in these patients are produced predominantly in the epithelial tissues. Furthermore, the presence of t-PA and PAI-2 seems to be most pronounced in the areas likely to be subjected to bacterial assault.

Increasing expression of tissue plasminogen activator and plasminogen activator inhibitor type 2 in dog gingival tissues with progressive inflammation.

Urokinase and tissue-type plasminogen activators (u--PA and t--PA) are serine proteases that convert plasminogen into plasmin, which degrades matrix proteins and activates metalloproteinases. The PAs are balanced by specific inhibitors (PAI--1 and PAI--2). Local production of t--PA and PAI--2 was recently demonstrated in human gingival tissues. The aim now was to investigate the production and localization of t--PA and PAI--2 in gingival tissues from dogs in three well-defined periodontal conditions; clinically healthy gingiva, chronic gingivitis and an initial stage of ligature-induced loss of attachment. At the start of the experiment the gingiva showed clear signs of inflammation. Clinically healthy gingiva were obtained after 21 days period of intense oral hygiene. Attachment loss was induced by placing rubber ligatures around the neck of some teeth. Biopsies were taken from areas representing the different conditions and prepared for in situ hybridization and immunohistochemistry. In clinically healthy gingiva both t--PA mRNA and antigen were expressed in a thin outer layer of the sulcular and junctional epithelia. No t--PA signals or staining were seen in connective tissue. Both mRNA signaling and immunostaining for t--PA were stronger in chronic gingivitis. In areas with loss of attachment, t--PA mRNA as well as antigen were found in the sulcular and junctional epithelia to a similar degree as in gingivitis. Occasionally the connective tissue was involved, especially in connection with vessels. PAI--2 mRNA was seen in a thin outer layer of the sulcular and junctional epithelia in clinically healthy gingiva, but no signals were seen in connective tissue. PAI--2 antigen was found primarily in the outer layer of the sulcular and junctional epithelia. Some cells in the connective tissue were stained. In gingivitis, PAI--2 signals were mainly found in the same locations, but more intense and extending towards the connective tissue. Immunostaining was seen in the outer half of the sulcular and junctional epithelia as well as in the upper part of the connective tissue, close to the sulcular epithelium. In sites with loss of attachment, PAI--2 mRNA was found throughout the sulcular and junctional epithelia, as was the antigen, which stained intensely. No PAI--2 mRNA was seen in connective tissue; the antigen was found scattered, especially near vessels. This study shows that the expression of both t--PA and PAI--2 increases with experimental gingival inflammation in the dog, and furthermore, the two techniques demonstrate a strong correlation between the topographical distribution of the site of protein synthesis and the tissue location of the antigens for both t--PA and PAI--2. The distribution correlates well with previous findings in humans.

The plasminogen-activating system in gingival fluid from adults. An intra-individual study before and after treatment of gingivitis.

High concentrations of tissue plasminogen activator (t-PA) and placental type plasminogen activator inhibitor (PAI-2) have previously been found in gingival crevicular fluid (GCF) of adults and children. In the present study, intra-individual comparisons were made of the concentrations of t-PA, urokinase type plasminogen activator (u-PA), PAI-1, and PAI-2 in GCF from the same sites before and after periodontal treatment in eight healthy male volunteers aged 35-46 yr. The gingival state was assessed by exudate measurement, bleeding on standardized probing, and the gingival index of Löe & Silness 3 days before the start of the trial and on the day after completing a 21-day preventive program consisting of instruction and professional cleaning once a week. Eight sites per subject were selected for enzyme analyses, all showing improvement in gingival state during the period. Sampling of GCF at the start and at the end of the trial was done with small disks of Millipore-filter. t-PA and PAI-2 were analyzed with enzyme-linked immunosorbent assays with low method errors. The mean concentrations of t-PA were 0.73 mg/l before treatment and 0.49 mg/l after treatment. The mean concentrations of u-PA were 84.4 micrograms/l before treatment and 101.6 micrograms/l after treatment. PAI-1 was found in three subjects at the detection level. The mean PAI-2 concentrations were 2.19 mg/l before and 1.13 mg/l after treatment. The mean molar ratio PAs/PAI-2 was 0.47 before and 0.48 after treatment. This insignificant change implies a maintained proteolytic balance and indicates that PAI-2 is an important inhibitor of tissue proteolysis.

Tissue plasminogen activator (t-PA) and placental plasminogen activator inhibitor (PAI-2) in gingival fluid from 8-9-year-old children.

High concentrations of tissue plasminogen activator (t-PA) and placental type plasminogen activator inhibitor (PAI-2) have previously been found in gingival crevicular fluid (GCF) of adults. In the present study, the levels were examined in 16 children aged 8-9 yr. Sampling of GCF was performed with small disks of Millipore-filter. t-PA and PAI-2 were analyzed with enzyme-linked immunosorbent assays with low method errors. The mean concentration of t-PA was slightly higher than in adults, while the mean PAI-2-concentration was slightly lower. An intraindividual study comparing healthy and inflamed sites in the children showed slightly higher concentrations in GCF from inflamed sites. No change was observed in the balance between t-PA and PAI-2.